The present invention relates to a method and apparatus for DNA electrophoresis and detection.
Electrophoretic lanes are widely used for separating multi-component samples ranging from small inorganic ions to large biological molecules. DNA electrophoresis is commonly performed with polyacrylamide gel placed between two glass plates. In recent years, the method of capillary electrophoresis has been developed, which alleviates the dissipation of Joule heat and permits the application of higher voltage, thus speeding up the electrophoresis separation process. In capillary electrophoresis, a buffer-filled capillary is suspended between two reservoirs filled with a buffer liquid. An electric field is applied between the two ends of the capillary. The potential difference that generates the electric field is in the range of kilovolts. Multi-component samples are typically injected under the influence of an electrical field. The samples migrate under the influence of electric field, with components of the sample being electrophoretically separated. After the separation, the components are detected by a detector.
One of the important applications of electrophoretic separation is for DNA sequencing. The use of capillary electrophoresis has improved DNA sequencing rates. Part of the improvement in speed, however, was initially offset by the loss of the ability (inherent in slab gels) to accommodate multiple lanes in a single run. Highly multiplexed capillary electrophoresis, by making possible hundreds or even thousands of parallel sequencing runs, offers an attractive approach to overcoming the current throughput limitations of DNA sequencing instrumentation. Typically, an array of capillaries is held in a guide and the intake (cathode) ends of the capillaries are dipped into vials that contain samples. After the samples are taken in by the capillaries, the ends of the capillaries are removed from the sample vials and submerged in a buffer which can be in a common container or in separate vials.
The currently used multichannel electrophoretic arrays typically represent a coplanar arrangement of capillaries. This geometry has been chosen because of its convenience for detection, which is typically performed with the help of fluorescent tags (fluorophores) attached to the DNA fragments migrating along the electrophoretic lanes. The detection is typically effected by illuminating the lanes within a specially provided translucent portion near their anode end (the observation region) with a laser source that excites fluorescence. One of the common reasons for the conventional planar arrangement of the capillaries has been that it offers a straightforward way of positioning the photoreceiving matrix that detects the fluorescence from all lanes in parallel. Another common reason for the parallel arrangement of capillaries is due to the need for color resolution of different fluorescent markers, which is typically performed by spatially dispersing the emitted fluorescent radiation in the longitudinal (along the lanes) direction. The spatially dispersed radiation from all observation regions is then imaged onto a two-dimensional photoreceiving matrix, such as CCD or CMOS, using a high-aperture projection objective. Still another common reason for the parallel arrangement of capillaries is associated with the desire to illuminate all lanes at once with a laser beam, which propagates in the plane of the capillaries and at the same time transverse to their axes.
In recent years, several authors disclosed such multicapillary systems, see e.g., Quesada et al., xe2x80x9cMultiple capillary DNA sequencer that uses fiber-optic illumination and detectionxe2x80x9d, Electrophoresis, vol. 17, pp. 1841-1851 (1996). Moreover, multicapillary systems have been disclosed in which the capillaries themselves serve as light-guiding elements for the illumination beam, see, e.g., Yeung et al., xe2x80x9cMultiplexed capillary electrophoresis systemxe2x80x9d, U.S. Pat. No. 5,582,70 (1996) and Quesada et al., xe2x80x9cMulti-capillary optical waveguides for DNA sequencingxe2x80x9d, Electrophoresis, vol. 19, pp. 1415-1427 (1998).
Therefore, a need exists for a non-planar arrangement of multiple capillary electrophoretic lanes which provide miniaturization of the electrophoretic carrier and which will significantly reduce the cost of multiple-lane DNA sequencing machines. A further need exists for a method for manufacturing monolithic cassettes, including multiple capillary lanes and a method and apparatus for parallel detection of fluorescent markers passing through the observation regions in a non-planar arrangement of multiple electrophoretic lanes.
The present disclosure describes a non-planar arrangement of multiple capillary electrophoretic lanes, a technique for manufacturing monolithic cassettes, comprising such multiple capillary lanes and a method and apparatus for parallel detection of fluorescent markers passing through the observation regions in a non-planar arrangement of multiple electrophoretic lanes. The need for non-planar arrangement arises from the desire to miniaturize the electrophoretic carrier, which will significantly reduce the cost of multiple-lane DNA sequencing machines.
The present disclosure offers inventive solutions that circumvent all of the above-cited common reasons for choosing co-planar geometry of a multilane assembly. In the simplest embodiment, the photoreceiving matrix is arranged in a first plane inclined at an angle relative to the capillary axes, while the observation regions of different capillaries are arranged in a second plane which is also inclined at an angle relative to the capillary axes. For example, the first and second planes are parallel to each other inclined at 45 degrees relative to the capillary axes. The simultaneous illumination of multiple capillary lanes is effected by an array of modulated laser sources whose beams have a specially chosen spatial arrangement and direction relative to the capillary axes and to said first and second planes. Next, the need for spatial dispersion of fluorescent radiation into components corresponding to different fluorescent wavelengths is eliminated in accordance with the method for multicolor fluorescent detection recently disclosed by Gorfinkel et al., xe2x80x9cMethod and apparatus for identifying fluorophoresxe2x80x9d, U.S. Pat. No. 5,784,157 (1998). Further, the need for waveguiding the incident radiation in the inventive method is substantially eliminated by using tightly packed capillaries of small cross-section. In a preferred embodiment, the capillaries have a rectangular or square cross-section of less than about 100 xcexcm on the side. For example, a rectangular array of 96 such capillaries has an overall cross-section of less than 1 mm2. As many as one thousand capillary lanes can be accommodated in a monolithic array of square cross-section about 3xc3x973 mm. The present invention further discloses techniques for fabricating such multicapillary arrays. These techniques employ drawing a glass preform that has a pre-fabricated set of holes of desired shape (e.g., rectangular) and is similar to drawing hollow optical fibers or glass ferrules, see, e.g., MacChesney et al., xe2x80x9cMaterials development of optical fiberxe2x80x9d, Journal of the American Ceramic Society, vol. 73, pp. 3537-3556 (1990) and Anderson et al., xe2x80x9cOptical fiber connector comprising a glass ferrule, and method of making samexe2x80x9d, U.S. Pat. No. 5,598,496 (1997). In one preferred embodiment, the preform is prepared with multiple holes to draw a monolithic multicapillary structure. In another preferred embodiment, a multicapillary bundle is fabricated by gluing or soldering together a multiplicity of single capillaries.
Still another aspect of the present invention pertains to loading tightly packed monolithic capillaries. In one of the preferred embodiments, this is provided by matching the capillary array cross-section with a similar array of charging pins on a silicon chip. In another preferred embodiment, the capillary assembly, which is monolithic in the observation region near the anode, is made loose like a brush near the cathode end. A special fixture holder is further provided that fixes the loose cathode ends of capillaries in a desired pattern. In a preferred embodiment, the loose cathode ends of the capillaries are arranged in a pattern that matches the common 96 well plate widely used in the preparation of biological samples.